Regulation and Function of RAB7 in the pulmonary endothelium

Pulmonary arterial hypertension (PAH) has a 3-year mortality rate of up to 55%. The survival benefit of vasodilator therapy does not last beyond one year, indicating that pulmonary artery (PA) remodeling, rather than vasoconstriction, predicts long-term prognosis. New anti-proliferative therapies emphasize targeting PA remodeling in PAH. A better understanding is needed of how ECs create an abnormal microenvironment that promotes PA remodeling. This proposal aims to fill this crucial knowledge gap by elucidating a novel mechanism by which ECs profoundly impact other PA mural cells. The discovery that the endosomal GTPase RAB7 is a critical gatekeeper for lung EC and vascular function forms the foundation of this work. However, the molecular regulation of RAB7 expression in ECs in PAH and the mechanism by which endothelial RAB7 deficiency induces pulmonary hypertension (PH) remain unclear. Here, studies are proposed to address these two critical issues. Based on new supportive data, the unique paradigm is presented that hypoxia signaling represses endothelial RAB7 transcription in PAH, promoting PA remodeling and PH via an altered secretome. Exciting new data reveal MEIS2 as a new transcriptional inducer of RAB7 in ECs. Additionally, a previously unknown repression mechanism is shown where hypoxia-inducible factor (HIF)- 2α accelerates MEIS2 mRNA decay via microRNA (miR)-199a. Unbiased screening identified a new interaction between RAB7 and the RNA-binding protein cold shock domain containing E1 (CSDE1) in ECs. The loss of RAB7:CSDE1 interaction upregulated target mRNAs via CSDE1, subsequently increasing their levels in the secretome and promoting PH via the proliferation of PA smooth muscle cells (PASMCs) and PA adventitia fibroblasts (PAAFs). Notably, the FDA-approved CSDE1 modulator Clofoctol reduced experimental PH. These results led to the HYPOTHESIS: In PAH, the transcriptional repression of RAB7 in ECs arises from accelerated decay of MEIS2 mRNA via the HIF-2α→miR-199a pathway and leads to the CSDE1- dependent dysregulation of the endothelial secretome, which contributes to PH. This hypothesis will be tested with the two SPECIFIC AIMS: Aim 1. To determine the MEIS2-driven transcriptional regulation of RAB7 in ECs. Aim 2. To investigate the molecular mechanism of RAB7-dependent PA remodeling via CSDE1 in ECs. In vitro experiments using gene manipulation, co-culture experiments, in vivo rat and mouse experiments, and Omics approaches will be employed to test the hypothesis. This research will establish a new molecular model connecting RAB7 transcriptional regulation via the HIF-2α→miR-199a→MEIS2 axis to EC dysfunction and PAH. This approach will further elucidate for the first time the molecular link between RAB7 deficiency, mRNA stability, PASMC and PAAF growth, and PA remodeling by studying CSDE1 as a key regulator of the endothelial secretome. These results will fundamentally enhance the understanding of EC dysfunction in PAH and provide novel therapeutic opportunities.